The present invention relates to the molecular cloning of genes encoding transferrin receptor (TfR) proteins and, in particular, to the cloning of transferrin receptor genes from Moraxella (Branhamella) catarrhalis. 
Moraxella (Branhamella) catarrhalis bacteria are Gram-negative diplococcal pathogens which are carried asymptomatically in the healthy human respiratory tract. In recent years, M. catarrhalis has been recognized as an important causative agent of otitis media. In addition, M. catarrhalis has been associated with sinusitis, conjunctivitis, and urogenital infections, as well as with a number of inflammatory diseases of the lower respiratory tract in children and adults, including pneumonia, chronic bronchitis, tracheitis, and emphysema (refs. 1 to 8). (Throughout this application, various references are cited in parentheses to describe more fully the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). Occasionally, M. catarrhalis invades to cause septicaemia, arthritis, endocarditis, and meningitis (refs. 9 to 13).
Otitis media is one of the most common illnesses of early childhood and approximately 80% of all children suffer at least one middle ear infection before the age of three (ref. 14). Chronic otitis media has been associated with auditory and speech impairment in children, and in some cases, has been associated with learning disabilities. Conventional treatments for otitis media include antibiotic administration and surgical procedures, including tonsillectomies, adenoidectomies, and tympanocentesis. In the United States, treatment costs for otitis media are estimated to be between one and two billion dollars per year.
In otitis media cases, M. catarrhalis commonly is co-isolated from middle ear fluid along with Streptococcus pneumoniae and non-typable Haemophilus influenzae, which are believed to be responsible for 50% and 30% of otitis media infections, respectively. M. catarrhalis is believed to be responsible for approximately 20% of otitis media infections (ref. 15). Epidemiological reports indicate that the number of cases of otitis media attributable to M. catarrhalis is increasing, along with the number of antibiotic-resistant isolates of M. catarrhalis. Thus, prior to 1970, no xcex2-lactamase-producing M. catarrhalis isolates had been reported, but since the mid-seventies, an increasing number of xcex2-lactamase-expressing isolates have been detected. Recent surveys suggest that 75% of clinical isolates produce xcex2-lactamase (ref. 16, 26).
Iron is an essential nutrient for the growth of many bacteria. Several bacterial species, including M. catarrhalis, obtain iron from the host by using transferrin receptor proteins to capture transferrin. A number of bacteria including Neisseria meningitidis (ref. 17), N. gonorrhoeae (ref. 18), Haemophilus influenzae (ref. 19), as well as M. catarrhalis (ref. 20), produce outer membrane proteins which specifically bind human transferrin. The expression of these proteins is regulated by the amount of iron in the environment.
The two transferrin receptor proteins of M. catarrhalis, designated transferrin binding protein 1 (Tbp1) and transferrin binding protein 2 (Tbp2), have molecular weights of 115 kDa (Tbp1) and approximately 80 to 90 kDa (Tbp2). Unlike the transferrin receptor proteins of other bacteria which have an affinity for apotransferrin, the M. catarrhalis Tbp2 receptors have a preferred affinity for iron-saturated (i.e., ferri-) transferrin (ref. 21).
M. catarrhalis infection may lead to serious disease. It would be advantageous to provide a recombinant source of transferrin binding proteins as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents. The genes encoding transferrin binding proteins and fragments thereof are particularly desirable and useful in the specific identification and diagnosis of Moraxella and for immunization against disease caused by M. catarrhalis and for the generation of diagnostic reagents.
The present invention is directed towards the provision of purified and isolated nucleic acid molecules encoding a transferrin receptor of a strain of Moraxella or a fragment or an analog of the transferrin receptor protein. The nucleic acid molecules provided herein are useful for the specific detection of strains of Moraxella and for diagnosis of infection by Moraxella. The purified and isolated nucleic acid molecules provided herein, such as DNA, are also useful for expressing the tbp genes by recombinant DNA means for providing, in an economical manner, purified and isolated transferrin receptor proteins as well as subunits, fragments or analogs thereof. The transferrin receptor, subunits or fragments thereof or analogs thereof, as well as nucleic acid molecules encoding the same and vectors containing such nucleic acid molecules, are useful in immunogenic compositions for vaccinating against diseases caused by Moraxella, the diagnosis of infection by Moraxella and as tools for the generation of immunological reagents. Monoclonal antibodies or mono-specific antisera (antibodies) raised against the transferrin receptor protein, produced in accordance with aspects of the present invention, are useful for the diagnosis of infection by Moraxella, the specific detection of Moraxella (in, for example, in vitro and in vivo assays) and for the treatment of diseases caused by Moraxella.
In accordance with one aspect of the present invention, there is provided a purified and isolated nucleic acid molecule encoding a transferrin receptor protein of a strain of Moraxella, more particularly, a strain of M. catarrhalis, specifically M. catarrhalis strain 4223, Q8, R1, M35, 3 or LES1, or a fragment or an analog of the transferrin receptor protein.
In one preferred embodiment of the invention, the nucleic acid molecule may encode only the Tbp1 protein of the Moraxella strain or only the Tbp2 protein of the Moraxella strain. In another preferred embodiment of the invention, the nucleic acid may encode a fragment of the transferrin receptor protein of a strain of Moraxella having an amino acid sequence which is conserved.
In another aspect of the present invention, there is provided a purified and isolated nucleic acid molecule having a DNA sequence selected from the group consisting of (a) a DNA sequence as set out in FIG. 5, 6, 10, 11, 27, 31, 32 or 33 (SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 45, 47, 48, 50 or 52 or the complementary DNA sequence thereto; (b) a DNA sequence encoding an amino acid sequence as set out in FIG. 5, 6, 10, 11, 27, 31, 32 or 33 (SEQ ID NOS: 9, 10, 11, 12, 13, 14, 15, 16, 46, 49, 51 or 53 or the complementary DNA sequence thereto; and (c) a DNA sequence encoding a functional transferrin receptor protein of a strain of Moraxella, which may be a DNA sequence which hybridizes under stringent conditions to any one of the DNA sequences defined in (a) or (b). The DNA sequence defined in (c) may have at least about 90% sequence identity with any one of the DNA sequences defined in (a) and (b). The functional transferrin receptor protein of a strain of Moraxella encoded by the DNA sequence defined in (c) is the equivalent transferrin receptor protein from another strain of Moraxella.
In an additional aspect, the present invention includes a vector adapted for transformation of a host, comprising a nucleic acid molecule as provided herein and may have the characteristics of a nucleotide sequence contained within vectors LEM3-24, pLEM3, pLEM25, pLEM23, SLRD-A, DS-1698-1-1, DS-1754-1, pSLRD2, pSLRD3, pSLRD4 and pSLRD5.
The vector may be adapted for expression of the encoded transferrin receptor, fragments or analogs thereof, in a heterologous or homologous host, in either a lipidated or non-lipidated form. Accordingly, a further aspect of the present invention provides an expression vector adapted for transformation of a host comprising a nucleic acid molecule as provided herein and expression means operatively coupled to the nucleic acid molecule for expression by the host of the transferrin receptor protein or the fragment or analog of the transferrin receptor protein. In specific embodiments of this aspect of the invention, the nucleic acid molecule may encode substantially all the transferrin receptor protein, only the Tbp1 protein, only the Tbp2 protein of the Moraxella strain or fragments of the Tbp1 or Tbp2 proteins. The expression means may include a promoter and a nucleic acid portion encoding a leader sequence for secretion from the host of the transferrin receptor protein or the fragment or the analog of the transferrin receptor protein. The expression means also may include a nucleic acid portion encoding a lipidation signal for expression from the host of a lipidated form of the transferrin receptor protein or the fragment or the analog of the transferrin receptor protein. The host may be selected from, for example, Escherichia coli, Bordetella, Bacillus, Haemophilus, Moraxella, fungi, yeast or baculovirus and Semliki Forest virus expression systems may be used. In a particular embodiment, the plasmid adapted for expression of Tbp1 is pLEM29 and that for expression of Tbp2 is pLEM33. Further vectors include pLEM-37, SLRD35-A and SLRD-35-B.
In an additional aspect of the invention, there is provided a transformed host containing an expression vector as provided herein. The invention further includes a recombinant transferrin receptor protein or fragment or analog thereof of a strain of Moraxella producible by the transformed host.
Such recombinant transferrin receptor protein may be provided in substantially pure form according to a further aspect of the invention, which provides a method of forming a substantially pure recombinant transferrin receptor protein, which comprises growing the transformed host provided herein to express a transferrin receptor protein as inclusion bodies, purifying the inclusion bodies free from cellular material and soluble proteins, solubilizing transferrin receptor protein from the purified inclusion bodies, and purifying the transferrin receptor protein free from other solubilized materials. The substantially pure recombinant transferrin receptor protein may comprise Tbp1 alone, Tbp2 alone or a mixture thereof. The recombinant protein is generally at least about 70% pure, preferably at least about 90% pure.
Further aspects of the present invention, therefore, provide recombinantly-produced Tbp1 protein of a strain of Moraxella devoid of the Tbp2 protein of the Moraxella strain and any other protein of the Moraxella strain and recombinantly-produced Tbp2 protein of a strain of Moraxella devoid of the Tbp1 protein of the Moraxella strain and any other protein of the Moraxella strain. The Moraxella strain may be M. catarrhalis 4223 strain, M. catarrhalis Q8 strain or M. catarrhalis R1 strain, M. catarrhalis M35 strain, M. catarrhalis 3 strain or M. catarrhalis LES1 strain.
In accordance with another aspect of the invention, an immunogenic composition is provided which comprises at least one active component selected from at least one nucleic acid molecule as provided herein and at least one recombinant protein as provided herein, and a pharmaceutically acceptable carrier therefor or vector therefor. The at least one active component produces an immune response when administered to a host.
The immunogenic compositions provided herein may be formulated as vaccines for in vivo administration to a host. For such purpose, the compositions may be formulated as a microparticle, capsule, ISCOM (immunostimulatory complex) or liposome preparation. The immunogenic composition may be provided in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces. The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant or at least one cytokine. Suitable adjuvants for use in the present invention include (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein. Advantageous combinations of adjuvants are described in copending U.S. patent applications Ser. Nos. 08/261,194 filed Jun. 16, 1994 and Ser. No. 08/483,856, filed Jun. 7, 1995, assigned to the assignee hereof and the disclosures of which are incorporated herein by reference thereto (WO 95/34308).
In accordance with another aspect of the invention, there is provided a method for generating an immune response in a host, comprising the step of administering to a susceptible host, such as a human, an effective amount of the immunogenic composition provided herein. The immune response may be a humoral or a cell-mediated immune response and may provide protection against disease caused by Moraxella. Hosts in which protection against disease may be conferred include primates, including humans.
In a further aspect, there is provided a live vector for delivery of transferrin receptor to a host, comprising a vector containing the nucleic acid molecule as described above. The vector may be selected from Salmonella, BCG, adenovirus, poxvirus, vaccinia and poliovirus.
The nucleic acid molecules provided herein are useful in diagnostic applications. Accordingly, in a further aspect of the invention, there is provided a method of determining the presence, in a sample, of nucleic acid encoding a transferrin receptor protein of a strain of Moraxella, comprising the steps of:
(a) contacting the sample with a nucleic acid molecule as provided herein to produce duplexes comprising the nucleic acid molecule and any nucleic acid molecule encoding the transferrin receptor protein of a strain of Moraxella present in the sample and specifically hybridizable therewith; and
(b) determining the production of the duplexes.
In addition, the present invention provides a diagnostic kit for determining the presence, in a sample, of nucleic acid encoding a transferrin receptor protein of a strain of Moraxella, comprising:
(a) a nucleic acid molecule as provided herein;
(b) means for contacting the nucleic acid molecule with the sample to produce duplexes comprising the nucleic acid molecule and any such nucleic acid present in the sample and hybridizable with the nucleic acid molecule; and
(c) means for determining production of the duplexes.
The invention further includes the use of the nucleic acid molecules and proteins provided herein as medicines. The invention additionally includes the use of the nucleic acid molecules and proteins provided herein in the manufacture of medicaments for protection against infection by strains of Moraxella.
Advantages of the present invention include:
an isolated and purified nucleic acid molecule encoding a transferrin receptor protein of a strain of Moraxella or a fragment or an analog of the transferrin receptor protein;
recombinantly-produced transferrin receptor proteins, including Tbp1 and Tbp2, free from each other and other Moraxella proteins; and
diagnostic kits and immunological reagents for specific identification of Moraxella.